DESCRIPTION: Cells do not simply reside within materials, they actively reengineer their microenvironments. The onset of cellular motility is characterized by dramatic degradation of the surrounding material due to attachment, traction and enzyme secretion. Just as cells modify their microenvironment, cells also receive cues from the material. The design of synthetic biomaterial scaffolds has aimed to recapitulate and harness this outside-in signaling to create materials that control cell motility. Understanding this phenomenon will advance the design of instructive materials that can spatially recruit and enhance encapsulated cell motility, the ?rst steps in the wound healing process. The proposed work will use highly engineered matrix metalloproteinase (MMP) degradable poly(ethylene glycol)-peptide hydrogel microenvironments to encapsulate human mesenchymal stem cells (hMSCs) and high spatio-temporal-modulus resolution microrheological characterization to measure dynamic scaffold remodeling. These results will enhance the understanding of how cells interact with and remodel materials prior to and during motility. Previous work used microrheological characterization to quantify the scaffold microenvironment during remodeling, identifying the time-dependent and spatial rheological properties of the scaffold. The degradation gradient measured around the hMSC shows greatest degradation furthest from the cell with stiff scaffold remaining directly around the cell. This suggests that the cell is inhibiting MMP scaffold degradation. We hypothesize that cells are secreting tissue inhibitors of metalloproteinase (TIMPs) to inhibit scaffold degradation to allow for attachment and spreading prior to complete degradation and accelerated motility. The proposed work will investigate the role of TIMPs in the degradation and remodeling of a well de?ned hydrogel scaffold environment. Speci?c Aim 1 will determine the role of TIMPs in matrix remodeling during 3D hMSC motility. This will be done by neutralizing TIMPs and measuring the resulting scaffold degradation. Simple models will be used to describe the degradation pro?le around the cell and pinpoint the type of degradation reaction occurring in the scaffold. Speci?c Aim 2 will determine if the physical microenvironment changes the role of TIMPs in scaffold degradation and cellular motility. The scaffold stiffness will be varied and MPT will be used to measure hMSC degradation. It is expected that as the material becomes stiffer cell-mediated degradation will become more aggressive and less MMP inhibition will occur. Collectively, the proposed work will identify the role of TIMPs in scaffold degradation and cellular motility determining whether the neutralization of TIMPs will lead to more aggressive cell-mediated degradation, resulting in accelerated motility that can be harnessed to spatially recruit cells and enhance motility.